For convenience, the large catalytic domains are designated by capital letters A and B, whereas the small t peptides are designated by lower case letters a and b, so that the wild-type A
Trang 1tail peptides in the oligomerization and secretory
trafficking of human acetylcholinesterase and
butyrylcholinesterase
Dong Liang1,2, Jean-Philippe Blouet1, Fernanda Borrega1, Suzanne Bon1and Jean Massoulie´1
1 Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supe´rieure, Paris, France
2 Key Laboratory of Brain Functional Genomics, MOE&STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, China
In vertebrates, butyrylcholinesterase (BChET) and the
T splice variant of acetylcholinesterase (AChET)
consist of a catalytic domain of approximately 500
residues, followed by C-terminal tail (t) peptides [1,2]
These peptides of 41 and 40 residues, respectively,
con-tain seven strictly conserved aromatic residues,
includ-ing three evenly spaced tryptophans, and a cysteine
located at position )4 from the C-terminus The
t peptide plays an important role in the biosynthesis of cholinesterases, particularly their folding and export For example, it has been shown that it induces the misfolding of a significant fraction of newly synthe-sized AChE polypeptides, and that this effect depends
on hydrophobicity since it was maintained when the aromatic residues were replaced by leucines The t pep-tide also reduces export, as indicated by the fact that
Keywords
acetylcholinesterase; butyrylcholinesterase;
cysteines; oligomers; secretion
Correspondence
J Massoulie´, Laboratoire de Neurobiologie,
CNRS UMR 8544, Ecole Normale
Supe´rieure, Paris, France
Fax: +33 1 44 32 38 87
Tel: +33 1 44 32 38 91
E-mail: jean.massoulie@biologie.ens.fr
(Received 13 August 2008, revised
25 September 2008, accepted
24 October 2008)
doi:10.1111/j.1742-4658.2008.06756.x
Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinester-ase that is predominant in mammalian brain and muscles (AChET) possess
a characteristic C-terminal tail (t) peptide This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba) We found that secretion was increased by deletion of the
t peptides, and that it was more efficient with a than with b The patterns
of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicat-ing a predominant influence of the catalytic domains However, addition of
a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetra-mers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cho-linesterases to form and secrete various oligomers Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations
Abbreviations
AchE T , T splice variant of acetylcholinesterase; BChE, butyrylcholinesterase; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide; Nbs2, 5,5¢-dithiobis(2-nitrobenzoic acid); PRAD, proline-rich attachment domains; t, tail.
Trang 2the ratio of secreted to cellular AChE was strongly
increased when it was deleted; this effect was
sup-pressed by mutation of the aromatic residues to
leucines [3–6]
However, the major function of the t peptides is that
they allow the assembly of tetramers of AChET[7] and
of BChET [8] and their association with the structural
proteins ColQ and PRiMA [9,10] These heteromeric
structures are based on a tight association between
four t peptides, also named tryptophan (W)
amphi-philic tetramerization domains, and the poly-proline
motifs, or proline-rich attachment domains (PRADs)
of ColQ and PRiMA [11–13] In addition, the BChE
tetramers that circulate in the blood plasma have
recently been shown to incorporate a similar
proline-rich peptide derived from the protein lamellipodin [14]
Crystallographic analyses of a complex of synthetic t
and PRAD peptides showed that four a-helical t
pep-tides form a coiled-coil around the PRAD, which is
arranged in a poly-proline II helix [15]
The assembly of cholinesterase homo-tetramers or
PRAD-associated tetramers is entirely conditioned by
the presence of a t peptide because truncated AChE
subunits lacking the t peptide only produce secreted
monomers [16] This peptide constitutes an
autono-mous interaction module, necessary and sufficient for
tetramerization and association with PRAD-containing
proteins, because addition of a t peptide at the
C-ter-minus of green fluorescent protein or alkaline
phos-phatase allowed the formation of tetramers associated
with an N-terminal fragment of ColQ [17] However,
the catalytic domains are also involved in quaternary
interactions that certainly participate in the formation
and stability of these oligomers In particular, the
tet-ramers are formed of two pairs of subunits, in which
a7,8 and a10helices from each subunit form a four
helix bundle, with a hydrophobic contact zone [16,18]
The respective contributions of the catalytic domains
and the t peptides in oligomers has not been evaluated
The formation of AChET tetramers associated with
PRAD-containing proteins is physiologically important
because it ensures their functional localization by ColQ
in the basal lamina at neuromuscular junctions [19], as
well as by PRiMA in cell membranes, particularly
in the brain [20] Similarly, the formation of BChET
tetramers conditions the secretion of this enzyme and
its stability in the bloodstream
Injection of AChE or BChE offers a very efficient
protection against poisoning by anti-cholinesterase
agents, such as organophosphorous pesticides, but
monomers and dimers are much more rapidly
elimi-nated than tetramers after injection in the circulation
[21–27] Although the half life of recombinant
enzymes, even monomers, in the bloodstream can be considerably increased by derivatization with polyeth-ylene glycol [28–33], it may be interesting to produce these enzymes as recombinant proteins in a stable tet-rameric form, which also present a greater thermal sta-bility than monomers or dimers [34]
Mutants lacking the cysteine located at)4 from the C-terminus do not form stable dimers, but can form tetramers, particularly in the presence of a PRAD-con-taining protein It is likely that transient dimerization occurs as a first step in the assembly of tetramers, either associated or not with a PRAD We recently found that addition of a second cysteine at an appro-priate position in the t peptide of Torpedo AChE greatly increased the formation and secretion of homo-tetramers [4] We therefore explored the possibility that mutations in the t peptides of human AChET and BChETmight induce their assembly into tetramers Because these two enzymes differ in their capacity to form oligomers, we investigated the respective roles of the catalytic domain and of the t peptides For this purpose, we constructed chimeric proteins, in which
we associated the catalytic domain of each enzyme with the t peptide of the other For convenience, the large catalytic domains are designated by capital letters (A and B), whereas the small t peptides are designated
by lower case letters (a and b), so that the wild-type AChE and BChE are Aa and Bb, and the chimeras are
Ab and Ba Comparisons of wild-type enzymes and chimeras, as well as of various mutants, show that both domains contribute critically to the oligomeriza-tion and to the efficiency of secreoligomeriza-tion
Results Exchange of t peptides between human AChE and BChE
The T variants of human AChE and human BChE are composed of a catalytic domain of approximately 500 residues, followed by small C-terminal t peptides of 40 and 41 residues, respectively In the present study, the catalytic domains are indicated by capital letters (A and B) and the C-terminal peptides by lower case letters (a and b), so that the wild-type enzymes are abbreviated as
Aa and Bb The C-terminal t peptides of human AChET (a) and BChET(b) are highly homologous, with 24 iden-tical residues (60%), including the seven aromatic resi-dues and the cysteine located at)4 from the C-terminus, being strictly conserved among all vertebrate cholines-terases (Fig 1A) However, they present significant dif-ferences, particularly between the residues immediately following the catalytic domain Some of the differences
Trang 3between the peptides a and b might be important for the
processing and the activity of AChE and BChE, notably
those involving charged residues, the presence in peptide
b of an additional tryptophan (W8) and the presence of
six instead of five residues between the aromatic-rich
region and the cysteine Both peptides are predicted to
form amphiphilic a helices, in which the aromatic
resi-dues are clustered in a sector of approximately 140
(Fig 1B)
To analyze the oligomerization properties due to the
t peptides of human AChE and BChE, we constructed
chimeras Ab and Ba in which we exchanged these
pep-tides; we also deleted the C-terminal peptides,
produc-ing the truncated enzymes A and B The different
constructs were expressed in transiently transfected
COS cells, and we analyzed the cellular and secreted
cholinesterase activities (Fig 2), as well as the
oligo-meric patterns, revealed by sedimentation profiles in
sucrose gradients (Fig 3)
The C-terminal t peptides do not influence the
catalytic activity of AChE and BChE
We examined the possible influence of the C-terminal
peptides on the AChE and BChE activities by
comparing the catalytic rates per active site The active
sites were titrated with the irreversible inhibitor
7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide
(DEPQ) (see Experimental procedures) The slopes of residual activity, plotted as a function of the amount
of DEPQ, were identical for A, Aa and Ab, with acetylthiocholine as substrate, as well as for B, Ba and Bb, using either acetylthiocholine or butyrylthio-choline as substrates Because of excess substrate inhi-bition, AChE presented a maximal rate for approximately 2 mm acetylthiocholine The rates of hydrolysis of acetylthiocholine and butyrylthiocholine (at 6 mm) by BChE were approximately 14% and 24% of the rate of hydrolysis of acetylthiocholine (at 2 mm) by AChE
Influence of the C-terminal t peptides on activity, secretion and oligomerization
As expected, the truncated mutants A and B, without
t peptides, produced only monomers, sedimenting around 4S (not shown) The levels of cellular activity were lower for these mutants than for the wild-types but secretion was increased (Fig 2A,B), in agreement with our previous conclusions that t peptides induce a partial misfolding of the polypeptides, as well as an intracellular degradation of a fraction of active subunits [3]
Cells expressing wild-type human AChE (Aa) secreted approximately 15% of their content per hour and produced mostly monomers and dimers, with a small proportion of tetramers (less than 10% of the
A
B
Fig 1 Structures of AChE and BChE t peptides (A) Sequences of the C-terminal t peptides of human AChE and BChE These peptides (a and b) are encoded by 3¢ exons from the cholinesterase genes; in the present study, we numbered from their first residue The seven aromatic residues, which are conserved in all vertebrate cholinesterases, are shown in blue; acidic residues are shown in red and basic resi-dues in green; the cysteines are indicated by orange arrowheads and the resiresi-dues that have been mutated to cysteines in the present study are underlined Residues that differ between peptides a and b and were mutated in b are indicated by vertical lines above the sequence (those which were mutated as a group are joined by an horizontal line) (B) En face view of the a helices formed by the N-terminal regions
of peptides a and b Colours are as in (A), except that cysteines are shown in orange and residues that were mutated to cysteines are indi-cated by orange circles.
Trang 4secreted activity) For human BChE (Bb), the rate of
secretion was only approximately 5% of the cellular
content per hour This enzyme formed a higher
pro-portion of oligomers, mostly dimers in the cells, and
tetramers represented approximately 30% of the
secreted enzyme, together with comparable proportions
of dimers and monomers (Fig 3A)
The fact that Bb forms a higher proportion of olig-omers, but is less efficiently secreted than Aa, is quite paradoxical because secretion generally increases with the degree of oligomerization Clearly, the assembly
of tetramers is not restricted by the fact that each BChE subunit possesses nine N-linked glycans [35], whereas AChE has only four This was confirmed by
0 50 100
150 200
- A-Aa
Aa S19C
Aa S38C -Ab
Ab SSVGL
Ab N19C
Ab N19C N18S
Ab N19C MD22VH
Ab N19C N18S MD22VH
- B-Ba
Ba S19C -Bb
Bb SSVGL
Bb A12C
Bb H15C
Bb N19C
Bb N26C
Bb S37C
Bb N19C N18S
Bb N19C MD22VH
Bb N19C N18S MD22VH
-Cellular activity
A
B
Secreted activity
0 1 2 3 4 5 6
A- Aa
Aa S19C Aa S38C
B- Ba
Bb A12C Bb H15C Bb N19C Bb D23C Bb N26C Bb S37C
Ratio of secreted to cellular acti
Fig 2 Cellular and secreted activities produced by human AChE, BChE and mutants used in the present study (A) Cellular and secreted activities A and B represent AChE and BChE from which the t peptides were deleted; Aa and Bb represent the wild-type enzymes with their t peptides, Ab and Ba represent chimeras in which the t peptides were exchanged; mutations in the t peptides are indicated For each mutant, the cellular and secreted activities are shown by bars to the left and the right AChE and BChE activities were determined by the Ellman assay with acetylthiocholine and butyrylthiocholine as substrates, respectively: AChE activities are indicated as grey bars and BChE activities as hatched bars AChE and BChE activities were normalized to the wild-type enzymes (Aa and Bb, respectively) (B) Ratio
of secreted to cellular activity Note that the double mutation M22V ⁄ D23H is abbreviated as MD22VH.
Trang 5the fact that mutants lacking some of the
N-glycosyl-ation sites, which were provided by O Lockridge [36],
did not produce a higher proportion of tetramers
(not shown)
For the chimeras Ab and Ba, the rates of secretion
were approximately 5% and 15% of the cellular
con-tent per hour, respectively, and therefore appeared to
be mainly determined by the t peptides By contrast,
Fig 3B shows that the sedimentation profiles were
very similar for Aa and Ab, and for Ba and Bb, except that the BChE species sedimented faster than their AChE counterparts, in agreement with the higher mass of BChE subunits [37] This indicates a predominant influence of the catalytic domain on oligomerization
Role of the C-terminal cysteine Mutation of the cysteine located at )4 from the C-ter-minus to a serine in the a or b peptides suppressed the formation of Aa or Bb dimers, but not the production
of a small proportion of tetramers (not shown) These mutations increased the ratio of secreted to cellular activity in both cases (Fig 2) However, in the case of
Bb, the cellular activity was decreased and secretion was increased, suggesting that the presence of this cys-teine retains the enzyme intracellularly In case of Aa, the cellular activity of AChE was also decreased by approximately 50% but secretion was not modified, suggesting that degradation was increased by suppres-sion of the cysteine
Thus, it appears that the effect of a C-terminal cysteine on the trafficking of cholinesterase in the secretory pathway largely depends on the nature of the preceding catalytic domain
Oligomerization might be affected by the distance between the aromatic core and the C-terminal cysteine, which forms inter-catenary disulfide bonds There are five residues between Y31 and this cysteine in peptide a, and six in peptide b, because of an additional residue, T32 To evaluate the possible influence of this residue
on oligomerization, we deleted T32 in Bb and we mutated peptides a (CSDL to SCDL) and b (SCVGL to CSVGL), to modify the number of residues between the cysteine and the aromatic core We found that these mutations had no effect on either the levels of cellular and secreted activities, or on the distribution of oligo-meric forms (not shown) Similarly, these mutations did not modify the secretion or the oligomerization of mutants possessing a cysteine at position 19 (see below) Thus, the addition or subtraction of one residue in the interval between the aromatic residues and the cysteine had no influence, suggesting that this peptidic segment represents a flexible spacer, in agreement with previous studies on Torpedo AChE [38]
Role of cysteines in oligomerization – effects of introducing an additional cysteine
In a previous study, we found that mutating residue
19 in the t peptide of Torpedo AChE considerably increased the production and secretion of tetramers
Cell extract
Medium
G1 G2
G4
G1
G2 G4
G1
G2
G4
G1
G2 G4 G3 G6
G1
G2
G4
G1
G2 G4
G1 G2
G4
G1
G2 G4
Aa
Bb
Ba
Ab
Aa
Sedimentation coefficients
10
Wild-type t peptides
19C
Bb
Ba
Ab 19C
19C
19C
Fig 3 Sedimentation profiles indicating the proportions of
oligo-meric forms produced by AChE, BChE, chimeras and mutants (A)
Left panels: Aa, Ab, Ba, Bb (B) Right panels: mutants containing a
cysteine at position 19 (S19C in peptide a, N19C in peptide b) The
profiles corresponding to cell extracts are shown with filled
sym-bols (d, AChE; , BChE) and a continuous line, and those
corre-sponding to the medium with empty symbols (s, AChE; h, BChE)
and a dashed line The peaks corresponding to tetramers, dimers
and monomers are indicated as G4, G2and G1, respectively Note
that the molecular forms of BChE and its mutants sediment slightly
faster than the corresponding AChE molecular forms.
Trang 6[4] We therefore introduced similar mutations in a
and b, and analyzed the resulting activities and
molecular forms produced by expressing the four
cholinesterase combinations in COS cells This
mutation did not modify the level of secretion for
Aa19C, increased it for Ba19C, and decreased it for
Ab19C and Bb19C (Fig 2) The fact that the cellular
activity was unchanged or decreased, whereas
sec-retion was decreased, indicates that the N19C
mutation in peptide b induced an intracellular
degra-dation of Ab19C and Bb19C As observed in the
pre-ceding section, the ratio of secreted to cellular
activity again appeared to depend essentially on the
t peptides: it was much higher for Aa19C and Ba19C
than for Ab19C and Bb19C The 19C mutations
enhanced the difference between the two peptides
because the secreted⁄ cellular ratio was increased with
peptide a19C compared to a and decreased with
pep-tide b19C compared to b
By contrast to the oligomeric patterns obtained
without a cysteine at position 19, we observed a
much stronger similarity between enzymes possessing
the same C-terminal peptide (a19C or b19C) than
between those possessing the same catalytic domain
(Fig 3B) Thus, mutation S19C in a19C strongly
increased the proportion of tetramers, which became
the predominant secreted species for both Aa19C and
Ba19C: these mutants produced very similar patterns
of molecular forms The effect of mutation N19C in
peptide b19C had little effect on the distribution of
secreted molecular forms of Bb19C In the case of
Ab19C, the effect was more complex: the cells
con-tained mostly 4S monomers but secreted a variety of
oligomers, mostly monomers, dimers, trimers,
tetra-mers and hexatetra-mers (see below) The fact that the
oligomeric forms were very low or undetectable in
the cells suggests that they were secreted very rapidly
after their assembly The results were identical when
the 19C mutations were combined with mutations
that modified the distance between the C-terminal
cysteine and the aromatic residues, as indicated above
(not shown)
Taken together, these results show that the t
pep-tides possessing a cysteine at position 19 had a
stron-ger effect on the secretability of cholinesterases than
wild-type t peptides, and exerted a dominant influence
on oligomerization
Effects of introducing cysteines at different
positions in BChE
Our previous study of Torpedo AChE showed that the
pattern of oligomerization depended critically on
the position at which a cysteine was introduced in the
t peptide [4] Because the presence of a cysteine induced tetramerization at position 19 of peptide a, but not at position 19 of peptide b, we explored the effects of cysteines at other positions in BChE We mutated residues that, similar to N19, are located within the aromatic-rich segment of peptide b, but are oriented in the opposite sector of the a helix, produc-ing mutants A12C, H15C, D23C and N26C (Fig 1B)
We also added a second cysteine near the C-terminus (S37C), changing the C-terminal peptide from SCVGL
to CCVGL
These mutations had little effect on the cellular or secreted activities compared to wild-type BChE, except that the secreted⁄ cellular ratio presented a minimum with a cysteine at position 19, and was notably increased in the mutant possessing two C-terminal cysteines (S37C) As shown in Fig 4, the sedimenta-tion profiles of cellular enzyme varied mostly in the proportions of monomers and dimers, whereas tetra-mers remained low The ratio of ditetra-mers to monotetra-mers was markedly increased with cysteines in the N-termi-nal region of peptide b: b12C and even more for b15C
We previously reported a similar observation in the case of mutants of Torpedo AChE [4] The proportion
of tetramers was higher in the medium, and was maxi-mal with mutation N19C Therefore, position 19 appears to be the most favorable for tetramerization,
as previously observed in the case of Torpedo AChE
Is the difference between oligomerization and secretion caused by individual residues that differ between peptides a and b?
Because a and b peptides only differ at a few positions,
we introduced point mutations to reduce these differ-ences We made these mutations in Ab19C because the level of activity, secretion and molecular forms of this mutant were strikingly different from those of Aa19C (Fig 1B) We thus mutated the first three residues of peptide b (GNI) as a group; W8 and E9 together and separately; G13, N18, M22 and D23 together and sepa-rately; and N29 and D30 together, replacing these residues by the corresponding ones in a We also mutated KES to QDR, and VG to DL at the C-termi-nus We observed no marked effect of any of these mutations on the cellular or secreted activities, except that mutation W8R increased both cellular activity and secretion, in agreement with the notion that aromatic residues induce degradation of AChE through an endo-plasmic reticulum associated degradation process [3]
In all these mutants, the cellular extracts contained only a trace of tetramers, as observed for Ab and
Trang 7Ab19C (Fig 5) The sedimentation profiles of the secreted enzyme were similar to those obtained with
Ab19C, except that the proportion of tetramers (G4) was somewhat increased with N18S The M22V muta-tion mostly increased the 13.5S species, and the D23H mutation did not increase G4by itself, but their combi-nation, M22V⁄ D23H, induced a significant increase in the proportion of secreted tetramers
Hoping to obtain a higher yield of secreted tetra-mers, we then combined the N18S and M22D⁄ D23H mutations in Ab19C The combination of mutations N18S, N19C, M22D and D23H, abbreviated asS, pro-duced the highest proportion of secreted G4 tetramers and the highest secreted⁄ cellular activity ratio Because these mutations appear to favor the production of tetramers with the b peptide, we introduced them, separately and together, in Bb19C However, the resulting BbS mutant did not produce a higher proportion of tetramers than Bb19C(Fig 5)
Stokes radius and mass of oligomers
We wished to further characterize the oligomers of Ab19C and other mutants, some of which sedimented faster than tetramers, at 12.3S and 13.5S Because cholinesterase oligomers may be associated with elongated proteins such as collagen ColQ, their mass cannot be simply deduced from their sedimentation coefficient, but rather from a combination of their Stokes radius and sedimen-tation coefficient [39] We therefore used gel filtration chromatography to determine the Stokes radius of oligo-mers secreted by the mutant AbN19C-N29D-D30H, which was chosen because it produces the complete variety of
Ab oligomers (Fig 6A) The major oligomers were iso-lated from gradient fractions By comparison with the standard proteins b-galactosidase and alkaline phospha-tase, we obtained Stokes radii values, as indicated in Fig 6B We then determined the mass of these oligomers, assuming that it is proportional to the product of the sed-imentation coefficient and Stokes radius, as expected for proteins of similar density The values thus obtained indi-cated a globular structure because the mass was in fact proportional to S3⁄ 2 This relationship allowed us to determined the mass of the minor components, sediment-ing at 8.5S and 12.3S (Fig 6A) Figure 6C shows that
Cell extract Medium
G1
G 2
G 4
G 1
G 2
G 4
G1
G 2
G 4
G 1
G 2
G 4
G 1
G 2
G 4
G 1
G 2
G 4
Bb A12C
Sedimentation coefficients
5 10 15
Bb H15C
Bb N19C
Bb D23C
Bb N26C
Bb S37C
Fig 4 Sedimentation profiles of mutants of human BChE (Bb) with cysteines at positions 12, 15, 19, 23 and 26 The profiles obtained for Bb19C, also shown in Fig 3, are repeated here for comparison with the other mutants The symbols are as in Fig 3 Tetramers, dimers and monomers are indicated as G 4 , G 2 and G 1 , respectively The mutations replacing various residues by cysteines
in the C-terminal peptide are indicated.
Trang 8the masses of the six observed oligomers represent
multi-ples of the smaller one, demonstrating that they represent
monomers (G1), dimers (G2), trimers (G3), tetramers
(G4), pentamers (G5) and hexamers (G6)
Trimers, pentamers and hexamers were only formed
with an additional cysteine (S19C) Thus, mutants of
Ab can associate into these different multimers,
illustrating the versatility of associations between
t peptides possessing a cysteine at position 19 As noted above, most of these oligomers were observed in the medium but not in cells By contrast, BChE only formed monomers, dimers and tetramers
Discussion The C-terminal t peptides do not influence cholinesterase activity
The catalytic domain of cholinesterases is associated with two major types of C-terminal peptides: the
h peptides contain a signal for the post-translational addition of a glycolipid anchor and cysteines that allow the formation of disulfide-linked dimers, and the
t peptides allow the formation of a variety of oligo-mers These peptides are not required for catalytic activity because truncated enzymes, which are reduced
to their catalytic domains, are fully active Previous studies showed that oligomers of AChETsubunits pos-sessed the same turnover rate per site, but this did not exclude a possible influence of the nature of C-terminal peptides To examine this question, we titrated the active sites of truncated, wild-type and chimeric cho-linesterases with the irreversible inhibitor DEPQ, and compared their activities with the substrates acetylthio-choline and butyrylthioacetylthio-choline We found that the cat-alytic rate per active site only depends on the catcat-alytic domain: it was identical for truncated enzymes (A or B) and with enzymes possessing either a or b C-termi-nal peptides, in agreement with previous studies [40] showing that the variants AChET, AChERand a trun-cated mutant possessed the same Km value and excess substrate inhibition These results also show that the catalytic activity is not influenced by the oligomeric state of the enzymes, and thus justifies quantitative comparisons between the activities of the various mutants investigated in the present study
Effect of the C-terminal t peptides on folding and secretion
The t peptides of cholinesterases form amphiphilic
a helices with a sector containing their seven conserved aromatic residues This organization is critical for the association of cholinesterase tetramers with anchoring proteins containing a PRAD, and most probably also for the assembly of homomeric tetramers However,
we have previously shown that the presence of aro-matic residues in the t peptide reduces the production and secretion of AChE, at two distinct checkpoints [41] First, it induces a partial misfolding of newly synthesized polypeptides; this effect depends on the
G1
G2 G4 G3 G6
G1
G2 G4 G6
cell extract
medium
G1
G2 G4
G6
G1
G2
G4
G1
G2 G4
G3
G6
G1
G2
G4
G3
G6
G1
G2 G4
G3
Sedimentation coefficients 5
10
15
Ab N18S
N19C
Ab N19C
M22V
Ab N19C
D23H
Ab N19C
M22V D23H
Ab N18S N19C
M22V D23H
G1 G2 G6
G1
G2 G6
Bb N18S N19C
Bb N18S N19C M22V D23H
Bb N19C M22V D23H
5 10 15
Ab N19C
Fig 5 Effect of mutations suppressing differences between a and
b, on the distribution of oligomeric forms (A) Left panels and top
right panel: Ab 19C (B) Lower right panels: Bb 19C Sedimentation
patterns are shown as in Fig 3 The sedimentation profiles of the
Ab19Cmutant (top right panel) are repeated for comparison with
those obtained with additional mutations, which suppressed some
of the differences with peptide a Note that the effects of
muta-tions M22V and D23H are not additive.
Trang 9hydrophobic character of these residues because the
same effect was observed when they were replaced by
leucines [5] Second, they target a fraction of active
AChE subunits towards degradation by endoplasmic
reticulum associated degradation rather than secretion;
this effect depends on the presence of aromatic
resi-dues, rather than on hydrophobicity This quality
con-trol process may ensure that only correctly assembled
subunits are efficiently exported from the cells
The present results confirm that the production,
secretion and oligomerization of human AChE and
BChE are strongly influenced by their t peptides In
agreement with previous results, secretion was
consid-erably increased for both enzymes when the t peptides
were deleted Using chimeras in which these peptides
were exchanged (Ab, Ba), we further showed that the
ratio between secreted and cellular activities, which
may be taken as an index of secretability, was
essen-tially determined by the t peptide The rate of secretion
with the t peptide from AChE (a) was more than
two-fold higher than with the t peptide from BChE (b)
With modified t peptides possessing a cysteine near the
center of the aromatic cluster (S19C in a and N19C
in b), this difference became more than six-fold
The respective roles of the catalytic domains
and t peptides in oligomerization
Although the truncated A and B mutants only
pro-duced monomers, the Aa, Ab, Ba and Bb enzymes all
formed oligomers, including tetramers Because these tetramers were obtained without co-expression with a PRAD-containing protein, they most probably repre-sent homotetramers, in which the four t peptides may form a coiled coil complex with all aromatic residues oriented inwards, but without a central PRAD This hypothesis is supported by the fact that, although the presence of a PRAD only induces the assembly of tet-ramers, expression of some mutants without a PRAD produces tetramers together with other oligomers, including molecular forms sedimenting as trimers, pen-tamers and hexamers The odd-numbered complexes are not likely to represent heteromeric associations containing other proteins because they only occur with some Ab mutants with an added cysteine, and their masses correspond exactly to those expected for multi-ples of AChE subunits Because the formation of these unusual oligomers appears to depend strictly on the presence of an additional cysteine, they are probably stabilized by a network of inter-catenary disulfide bonds, linking all subunits together
The Ab19C chimera formed all oligomeric forms from monomers to hexamers, illustrating the versatility
of oligomeric associations based on the t peptide, in association with the catalytic domain It should be noted that hexamers have been observed in transfected COS cells expressing wild-type rat AChE, and appeared as a transient mode of association, which could be dissociated into monomers, dimers and tetra-mers (e.g in the presence of Triton X-100) [7] By
Ab N19C
N29D-D30H
secreted activity
G 1
G 2
G 3
G 4
G 5
G 6
Sedimentation coefficients
5 10
15
G 6
Elution coefficient (Ve-Vo)/(Vt-Vo)
-galactosidase Alkaline phosphatase
G 4 G 2
G 1
0 100 200 300 400 500
4.3 S 6.5 S
10.5 S
13.5 S
8.5 S
12.3 S
Numbers of subunits
G 6
G 5
G 4
G 3
G 2
G 1
Fig 6 Determination of the Stokes radius and mass of AChE B oligomers (A) Oligomers were isolated from sucrose gradients of medium from cells expressing the Ab 19C-29D-30H mutant The profile of cellular activity was identical to that shown in Fig 3B for the Ab 19C mutant (B) Elution of oligomers in gel filtration chromatography The elution parameters were defined as Ke= (Ve– Vo) ⁄ (V t – Vo), where Vo corre-sponds to the exclusion volume (blue dextran) and V t is the total volume (potassium ferricyanide) The Stokes radii were determined from the linear relationship between the Stokes radius and the square root of [ )log (K e )] using the standards b-galactosidase (6.9 nm, 16S,
464 kDa) and alkaline phosphatase (3.3 nm, 6.1S; 87 kDa) (C) The masses of the different oligomers were determined by their proportional-ity to the product of the Stokes radius with the sedimentation coefficient The masses of the minor 8.5S and 12.3S species were deter-mined from the linear relationship with S3⁄ 2, observed for the other oligomers The masses are found to be proportional to discrete degrees
of oligomerization, from 1 to 6, showing that the oligomers correspond to monomers (G1), dimers (G2), trimers (G3), tetramers (G4), penta-mers (G5) and hexamers (G6).
Trang 10contrast, Bb19C only formed the classical monomers,
dimers and tetramers, possibly because of steric
constraints due to the catalytic domain or to its
associ-ated N-glycans
Although the nature and proportions of oligomers
depended on the presence of the t peptides and their
cysteines, the catalytic domains also influenced the
oligomerization patterns The cellular and secreted
oligomers formed by Aa and Ab were very similar, as
well as those formed by Ba and Bb, suggesting a
pre-dominant influence of the catalytic domains on
oligo-merization This may be due in part to the difference
in N-glycosylation of AChE and BChE, which carry
four and nine N-glycan chains, respectively [35]; we
therefore compared the oligomeric patterns of
wild-type BChE and mutants in which part of the
N-glycosylation sites were mutated [36], but observed
no difference (not shown) The relative influence of the
C-terminal t peptide appeared to be strongly increased
when a cysteine was added at position 19 because the
patterns obtained for Aa and Ba were almost the same,
except for a shift in the sedimentation coefficients,
which are higher for BChE than for AChE
By contrast to Aa and Ab, oligomers of Ba and Bb
represented a significant proportion of cellular activity,
indicating that AChE oligomers were secreted more
rapidly after assembly than BChE oligomers It is
remarkable that the Ab19C oligomers were observed in
the medium but not in cell extracts This could be
related to the presence of the peptide b19C which
reduces secretion but may be masked in the oligomers
Thus, both the catalytic domain and the C-terminal
t peptides contribute to the control of oligomerization
and secretion, in a complex interplay
Origin and significance of the difference between
the t peptides of AChE and BChE
The a and b peptides present a considerable sequence
similarity, with 60% identical residues, including the
seven aromatic residues and the cysteine, which play a
key role in the interaction properties of the t peptides
In addition, both peptides are predicted to possess the
same tendency to form amphiphilic a helices It was
therefore unexpected to observe a strong difference in
their influence on the oligomerization and secretion of
AChE and BChE We tried to assign this difference
to some of the residues that distinguish the a and b
peptides Because oligomerization also depends on the
catalytic domains, as indicated by the difference
between the molecular forms produced by Ab19C and
Bb19C, the linkage between the two domains might well
play a crucial role in the quaternary associations of
the cholinesterase subunits The first three residues of peptides a and b are indeed different, but their replace-ment in Ab19C (GNI to DTL) had little effect on either secretion or oligomerization It is also noteworthy that the effects of the combined mutations M22D⁄ V23H could not be simply accounted for by the effects of the separate mutations M22V and D23V This suggests that the secretory trafficking of molecules containing peptides a and b depends on global properties of the peptides rather than on individual residues
AChE and BChE are expressed differentially during embryogenesis [42–44] They appear to play distinct roles, which may be based on their catalytic activity, but also on protein–protein interactions [45], because their catalytic domain is homologous to adhesion pro-teins such as neuroligin [46,47] For example, AChE may be involved in neurite extension during brain development [40,48,49] Both catalytic and noncatalytic functions clearly require appropriate oligomeric orga-nization and localization and therefore depend on the C-terminal t peptides, which may be directly involved
in distinct interactions with partner proteins
The two cholinesterases present a complex relation-ship with the development of Alzheimer’s disease, which may be partly related to their C-terminal t peptides Both AChE and BChE are associated with senile pla-ques in Alzheimer’s disease [50] but they appear to play antagonistic roles: AChE promotes amyloid aggregation and increases the neurotoxicity of the Ab peptide
in vitro, suggesting that it may participate in the patho-genesis of the disease [51,52]; this appears to depend on interactions of Ab peptides with the peripheral site of AChE and not on its C-terminal t peptide, which has no effect on Ab aggregation [53] By contrast, the C-termi-nal t peptide of BChE (peptide b) was found to reduce
Ab aggregation, possibly because of the presence of its additional tryptophan (W8) located opposite to the aro-matic cluster of the amphiphilic helix (Fig 1B), so that BChE might have a protective effect against Alzheimer’s disease [53,54] In this respect, it is worth recalling that, although the human AChE t peptide (here termed peptide a) is organized as an a helix, its AEFHRWS-SYMVHWK fragment, which resembles a portion of the amyloid Ab peptide (AEFRHDSGYEVHHQK), was found to organize into b sheets and to form fibrils;
by contrast, the homologous fragment from BChE
property [55–57]
AChE and BChE probably arose from a gene dupli-cation in the lineage of vertebrates and it is remarkable that sequence differences between their t peptides are strongly conserved, suggesting that they correspond to distinct molecular interactions and the oligomerization